scholarly journals CRISPR/dCas9 (D10A) -Mediated Hb CS Gene Editing and Identification of Genetically Modified Fibroblasts

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5754-5754
Author(s):  
Lian Yu ◽  
Wei Hao Wu ◽  
WANG Shunqing ◽  
Ken H. Young ◽  
Funeng Jiang
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Transfection Efficiency ◽  
Target Sequence ◽  
Gene Repair ◽  
Single Base ◽  
Editing Efficiency ◽  
Base Editing ◽  
Alpha Thalassemia

Background: Alpha-thalassemia (α-thalassemia) is an inherited hemolytic disease caused by complete or absent synthesis of α-globin chains due to alpha-globin chain synthesis disorders and is one of the most common genetic diseases in the world. At present, the most commonly used strategies in the gene therapy research of thalassemia, the nonhomologous end joining (NHEJ) apparatus, is only suitable for the study of beta thalassemia and sickle-type anemia. The DNA double strand breaks have a high risk to induce frameshift mutations, leading to more serious clinical consequences. Therefore, it is still necessary to study a safer genetic repair program for alpha-thalassemia. Methods: The donor we chose to repair was from a patient with a genotype of -SEA/αCSα, whose mutation is one of the most common thalassemia alleles in Longyan city, and the HBA1 gene is normal. Due to the deletion of a copy of the HBA2 gene, only one copy of the mutation needs to be repaired, making it a suitable candidate for the study of alpha thalassemia gene therapy. To minimize the risk of non-specific mutations introduced into the locus of interest, we used the single-base editing apparatus, dCas9 (dead Cas9), which only cleaves one strand of DNA and precisely modifies one base, effectively improve the specificity of target cleavage and reduce the risk of off-target. The recognition sequence on the homologous arm in the template was lengthened to reduce the risk of off-target.(see Figure 1 ) Result We used the pmaxGFP plasmid as a positive control for optimizing electroporation conditions. More than 80% transfection efficiency was obtained (see Figure 2 ) Fig 2 pmaxGFP transfection to verify the transfection efficiency of fibroblasts ( 100x ) To investigate the effectiveness of sgRNA and donor plasmids for mutated gene repair, we analyzed the repair efficiency of fibroblasts. Primers were designed near the repair site and PCR sequences were amplified by PCR . After amplification of the genomic fragments, BamH1 digestion assay was performed. Fig 3 PCR and Bam HI digested results using primers for the gene of interest, showed the gene editing efficiency was 4~10%. The sequencing results showed that three clonals of α- thalassaemia mutations were successfully repaired. Fig 4 Hb CS gene mutation target sequence repair and sequencing results The sequencing results of Fig. 5 show that no mutation was detected at the six potential off-target sites. Fig 5 off-target effect detection The use of base editing is limited by off-target activity and DNA delivery efficiency to cells. Filtration into fibroblasts by plasmid electroporation, CRISPR/Cas9-mediated editing efficiency is usually 3~10%, and we achieve a gene editing efficiency of 4~10% (Figure 4). Conclusions Our data show that the gene transfer of normal HBA2 gene by electroporation the single-base editor CRISPR/dCas9 (D10A) and donor into cells can successfully repair Hb CS mutations. Disclosures No relevant conflicts of interest to declare.

Reversal of Multidrug Resistance by Magnetic Fe3O4 Nanoparticle Copolymerizating Daunorubicin and mdr1-shRNA Expression Vetor in Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4829-4829
Author(s):  
Bao-An Chen ◽  
Peipei Mao ◽  
Jian Cheng ◽  
Feng Gao ◽  
Jia-Hua Ding ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cell Line ◽  
Recombinant Plasmid ◽  
Conflicts Of Interest ◽  
Transfection Efficiency ◽  
Resistance Index ◽  
Leukemic Cells ◽  
Target Sequence ◽  
Shrna Expression ◽  
Mdr 1

Abstract Abstract 4829 Object In many instances, Multidrug resistance (MDR) is mediated by increased expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe3O4 (MNP(Fe3O4)) and mdr1-shRNA Expression vetor.in K562/A02 leukemic cells. Methods To synthesis short hairpin RNA (shRNA)aiming divectly at the target sequence,we choice the 3491-3509,1539-1557and 3103-3121 nucleotide of mdr-1 mRNA as targets. Cloning in the plasmid vetor PGCSilencer-U6-neo-GFP, The recombinant plasmid vetors were called for PGY1-1,PGY1-2 and PGY1-3.The recombinant plasmid vetors were transfected into the cell 1ines K562/A02 by lipofection. After transfected 48 hours,the inhibition of mdr-1mRNA expression and the expression of P-gp was detected by realtime–PCR and Weston-blot, screening the recombinant plasmid vetor which has the most greatest mdr-1 gene inhibition ratio is PGY1-2.Analysis of the reveral ratio of multidrug resistance, the concentration of DNR and the content of mdr-1 gene and P-gp in K562/A02 cell line. Results The combination of daunorubicin (DNR) with either MNP(Fe3O4) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while MNP(Fe3O4) and PGY1-2 cotreatment can synergistically down regulation the expression of mdr-1 gene and the expression of P-gp(P<0.05). The transfection efficiency was 20%; the concentration of DNR in K562/A02 cell line was obviously elevated (P<0.05);the multidrug resistance index of K562/A02 cell line was obviously decreased (P<0.05). Conclusion MNP(Fe3O4) and PGY1-2 cotreatment can synergistically reveral multidrug resistance. Thus our in vitro data strongly suggests a potential clinical application of MNP(Fe3O4) and PGY1-2 combination on CML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

2020 ◽  
Author(s):  
Xi Xiang ◽  
Kunli Qu ◽  
Xue Liang ◽  
Xiaoguang Pan ◽  
Jun Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Editing ◽  
High Efficiency ◽  
Outcome Data ◽  
Massively Parallel ◽  
Human Embryonic Kidney Cells ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Sites ◽  
In Cells

AbstractThe CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

scholarly journals 216 Multiplex base editing of NK cell to enhance cancer immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A229-A229
Author(s):  
Minjing Wang ◽  
Mitchell Kluesner ◽  
Patricia Claudio Vázquez ◽  
Beau Webber ◽  
Branden Moriarity
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
High Efficiency ◽  
Cell Function ◽  
Nk Cell ◽  
Functional Assay ◽  
Single Base ◽  
Editing Efficiency ◽  
Base Editing

BackgroundNatural killer (NK) cells have many unique features that have gained attention in cancer immunotherapy. NK cells can kill in antigen independent and dependent fashion, can be used as an allogeneic product, and perform antibody-dependent cell-mediated cytotoxicity (ADCC). However, NK cell function is regulated by many activating and inhibitory receptors, which cancer cells take advantage of to avoid being killed by NK cells. NK cells are also known for their technical and biological challenges which result in low editing efficiencies, compared to T cells and other immune cells.MethodsBase editing (BE) is a CRISPR-Cas9 based genome editing technology that allows precise single base transitions. Previously, we reported a high efficiency method for multiplex engineering of T cells using BE and thus reasoned that applying similar concepts in NK cells may offer an opportunity to alter many genes simultaneously at higher efficiency through multiplex base editing. We thus selected a panel of genes bearing critical roles in NK cell function for immunotherapy, including inhibitory intracellular regulator AHR and CISH, inhibitory checkpoint receptor KLRG1, TIGIT, KLRC1, and PDCD1, and Fc receptor CD16A. CD16A is responsible for NK cell ADCC and is regulated via cleavage upon NK activation. Non-cleavable CD16A improves ADCC killing and can be achieved through single-base substitution with BE.ResultsUsing the adenosine BE (ABE8e), we achieved multiplex editing (6 genes) rates up to 99% and 95% editing/knockout at DNA and protein levels, respectively. Notably, we assessed for reduction in editing efficiency when additional genes were targeted and found no significant reduction in editing efficiencies when targeting up to 6 genes simultaneously. Moreover, functional evaluation of non-cleavable CD16A NK cells revealed up to 35% increase of cytotoxicity against Raji cells.ConclusionsWe were able to achieve high multiplex editing efficiency in primary human NK cells using ABE8eand there was no significant decrease of editing efficiency as the number of gene of interest increases, up to 6 genes in total. Functional assay confirmed increased NK cell cytotoxicity against tumor cells. Our end goal is to achieve high efficiency multiplex editing in CAR-expressing NK cells to further improve NK cell activity and toxicity for cancer immunotherapy.ReferenceWebber B, Lonetree C, Kluesner M, et al. Highly efficient multiplex human T cell engineering without double-strand breaks usingCas9 base editors. Nat Commun 2019;10:5222.

scholarly journals Homology-directed gene-editing approaches for hematopoietic stem and progenitor cell gene therapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar K. Azhagiri ◽  
Prathibha Babu ◽  
Vigneshwaran Venkatesan ◽  
Saravanabhavan Thangavel
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Target Cells ◽  
Gene Manipulation ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Base Editing ◽  
Stem And Progenitor Cells

AbstractThe advent of next-generation genome engineering tools like CRISPR-Cas9 has transformed the field of gene therapy, rendering targeted treatment for several incurable diseases. Hematopoietic stem and progenitor cells (HSPCs) continue to be the ideal target cells for gene manipulation due to their long-term repopulation potential. Among the gene manipulation strategies such as lentiviral gene augmentation, non-homologous end joining (NHEJ)-mediated gene editing, base editing and prime editing, only the homology-directed repair (HDR)-mediated gene editing provides the option of inserting a large transgene under its endogenous promoter or any desired locus. In addition, HDR-mediated gene editing can be applied for the gene knock-out, correction of point mutations and introduction of beneficial mutations. HSPC gene therapy studies involving lentiviral vectors and NHEJ-based gene-editing studies have exhibited substantial clinical progress. However, studies involving HDR-mediated HSPC gene editing have not yet progressed to the clinical testing. This suggests the existence of unique challenges in exploiting HDR pathway for HSPC gene therapy. Our review summarizes the mechanism, recent progresses, challenges, and the scope of HDR-based gene editing for the HSPC gene therapy.

Correction of Fanconi Anemia Mutation Using the Crispr/Cas9 System

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3622-3622
Author(s):  
Nozomu Kawashima ◽  
Yusuke Okuno ◽  
Yuko Sekiya ◽  
Xinan Wang ◽  
Atsushi Narita ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Point Mutation ◽  
Fanconi Anemia ◽  
Insertional Mutagenesis ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Genetically Engineered ◽  
A Cell ◽  
Fancd2 Monoubiquitination

Abstract Introduction Gene therapy has been developed for genetic diseases, either to restore normal function for loss-of-function mutations or to inhibit gain-of-function mutations. Gene addition using genetically engineered viral and plasmid vectors has successfully corrected cell pathophysiology resulting in the production of functional proteins. Therapeutic safety has been reinforced by the use of self-inactivating vectors; however, the potential risk of tumorigenesis raises concerns for insertional mutagenesis combined with acquired somatic mutations. Recent advances in gene editing using an RNA-guided endonuclease (RGEN), known as the CRISPR/Cas9 system, have opened a new frontier for the in situ correction of disease-associated mutations. Genomic DNA of cells harboring mutations can be excised and replaced with a DNA template for the functional gene sequence using homology-directed repair (HDR). The advantages of this repair include fewer off-target effects and a reduced risk of copy number changes compared with gene addition using vectors. Fanconi anemia (FA) is a syndrome of inherited bone marrow failure, characterized by the deficient regulation of DNA double-strand break repair. Clinical trials of gene therapy using viral vectors are still on-going with partial success; therefore, a new gene editing technique deserves attention. However, the feasibility of this approach in diseases with impaired HDR, such as FA, is unknown. Therefore, we used an RGEN to generate a cell line harboring a disease-causing point mutation in an FA-associated gene and elucidated the efficacy of restoring the mutation thereafter. Methods pSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and single guide RNAs (sgRNAs) of interest. The sgRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate sgRNAs were designed to be specific for the FANCC c.67delG:p.D23Ifs*23 mutation type (MT) or wild type (WT): gRNA#4, 5′-ATGGGATCAGGCTTCCACTT-3′ and gRNA#5, 5′-GAAGCTTTCTGTATGGGATC-3′ were specific for the WT sequence; whereas, gRNA M4, 5′-TATGGATCAGGCTTCCACTT-3′ and gRNA M5, 5′-AGAAGCTTTCTGTATGGATC-3′ were specific for the MT sequence. pCAG-EGxxFP, an EGFP-based reporter plasmid for the HDR that harbored the 500-bp target region of the WT or MT FANCC, was constructed for the gRNA selection. An HDR template construct was designed to incorporate a puromycin-resistant gene flanked by two loxP sites and two homologous arms containing the WT or MT sequence. HEK293T cells harboring the WT FANCC sequence were genetically edited by the above-mentioned plasmids. Results To validate an efficient and specific sgRNA for DNA double-strand breaks, we cotransfected pCAG-EGxxFP-FANCC WT or MT and pSpCas9(BB)-FANCC-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR and thus the efficacy and specificity of sequence-specific DNA excision, was observed 48 h later, and we determined that gRNA#4 and gRNA M4 were specific for the WT and MT sequences, respectively. To generate cells harboring the MT FANCC sequence, HEK293T cells were cotransfected with pSpCas9(BB)-FANCC-gRNA#4 and the HDR template plasmid harboring the MT FANCC. A cell harboring biallelic MT FANCC was selected by adding puromycin and single-cell cloning. The transient expression of Cre recombinase in this clone successfully deleted the drug-selection cassette, and 293T-FANCC c.67delG cells were established. This cell showed the loss of FANCD2 monoubiquitination, a hallmark of a deficient FA core complex. Next, the 293T-FANCC c.67delG cells were cotransfected with pSpCas9(BB)-FANCC-gRNA M4 and the HDR template with the WT FANCC. This restoration of the mutated FANCC sequence resulted in a high frequency of at least monoallelic correction and the restoration of FANCD2 monoubiquitination. Conclusions The feasibility of genome editing was demonstrated in cells harboring an FA mutation, which can be a foothold for future therapy using precision gene restoration in patients with impaired HDR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2037-2037
Author(s):  
Takenobu Nii ◽  
Hiroshi Kohara ◽  
Tomotoshi Marumoto ◽  
Tetsushi Sakuma ◽  
Takashi Yamamoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Single Cell ◽  
Genome Editing ◽  
Pluripotent Stem Cells ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Editing Efficiency ◽  
Cell State ◽  
Transfection Method

Abstract Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to self-renew indefinitely and differentiate into various cell types. hPSCs can differentiate into various stem or progenitor cell populations used for regenerative medicine and drug development. Newly developed genome editing technology has advanced the use of hPSCs for such purposes. However, to fully utilize hPSCs to achieve this goal, more efficient gene transfer methods under defined conditions are required. Development of efficient genome editing methods, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9), for use in hPSCs holds great promise in the fields of basic and clinical research. Among these methods, TALENs are more efficient and safer for use in hPSCs to achieve specific gene editing, as ZFNs had a low gene editing efficiency and CRISPR/Cas9 was accompanied by more severe off-target effects than TALENs. Electroporation is a widely used transfection method for hPSC genome editing; however, this method results in reduced cell viability and gene editing efficiency. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a culture method of hPSCs at a single-cell-state to reduce cell density, and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell-state were transfected using non-liposomal transfection reagents with plasmid DNA driven by the human elongation factor 1-alpha 1 (EF1α) promoter or mRNA encoding enhanced green fluorescent protein (eGFP). The proportion of eGFP+ cells considerably increased in single-cell-state cultures (DNA: 95.80 ± 2.51%, mRNA: 99.70 ± 0.10%). Moreover, most of the cells were viable (control: 93.10 ± 0.40%, DNA: 83.40 ± 2.03%, mRNA: 86.71 ± 0.19%). The mean fluorescence intensity (MFI) was approximately three-fold higher than that in cells transfected by electroporation (electroporation (EPN): 6631 ± 992; transfection (TFN): 17933 ± 1595). eGFP expression was detected by fluorescence microscopy until day seven post-transfection. Our results also demonstrate an inverse correlation between cell density and transfection efficiency. To test whether transfection using this method affected the "stemness" of hPSCs, we examined SSEA4 and NANOG expression in eGFP-transfected cells by flow cytometry analysis. The percentage of both SSEA4+ and NANOG+ cells was greater than 90%. Moreover, transplantation of eGFP-transfected cells into immunodeficient mice led to the formation of teratomas. These results strongly suggested that single-cell-state hPSC culture improved transfection efficiency without inducing differentiation or loss of pluripotency. Moreover, we used our efficient transfection method to edit the hPSC genome using TALENs. We constructed a Platinum TALEN driven by the EF1α promoter targeting the adenomatous polyposis coli (APC) gene and analyzed the efficiency of gene editing using the Cel-1 assay. Our efficient transfection method induced mutations more efficiently than electroporation (Transfection: 11.1 ± 1.38%, Electroporation: 3.2 ± 0.89). These results showed that TALENs increased gene editing efficiency in single-cell-state hPSC cultures. Overall, our efficient hPSC transfection method using single-cell-state culture provides an excellent experimental system to investigate the full potential of hPSCs. We expect that this method may contribute to the fields of hPSC-based regenerative medicine and drug discovery. Disclosures No relevant conflicts of interest to declare.

scholarly journals GenEditID: an open-access platform for the high-throughput identification of CRISPR edited cell clones

2019 ◽  
Author(s):  
Ying Xue ◽  
YC Loraine Tung ◽  
Rasmus Siersbaek ◽  
Anne Pajon ◽  
Chandra SR Chilamakuri ◽  
...  
Keyword(s):  
Open Access ◽  
Gene Targeting ◽  
Gene Editing ◽  
Data Visualisation ◽  
Single Base ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Proteomic Data ◽  
Cell Clones ◽  
Specialist Groups

ABSTRACTCRISPR-Cas9-based gene editing is a powerful tool to reveal genotype-phenotype relationships, but identifying cell clones carrying desired edits remains challenging. To address this issue we developed GenEditID, a flexible, open-access platform for sample tracking, analysis and integration of multiplexed deep sequencing and proteomic data, and intuitive plate-based data visualisation to facilitategeneedited cloneidentification. To demonstrate the scalability and sensitivity of this method, we identified KO clones in parallel from multiplexed targeting experiments, and optimised conditions for single base editing using homology directed repair. GenEditID enables non-specialist groups to expand their gene targeting efforts, facilitating the study of genetically complex human disease.

scholarly journals Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ugne Zekonyte ◽  
Sandra R. Bacman ◽  
Jeff Smith ◽  
Wendy Shoop ◽  
Claudia V. Pereira ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Therapy ◽  
Mitochondrial Dna ◽  
Base Pair ◽  
Gene Editing ◽  
Single Base ◽  
Dna Mutations ◽  
Large Size ◽  
Dna Editing

AbstractDiseases caused by heteroplasmic mitochondrial DNA mutations have no effective treatment or cure. In recent years, DNA editing enzymes were tested as tools to eliminate mutant mtDNA in heteroplasmic cells and tissues. Mitochondrial-targeted restriction endonucleases, ZFNs, and TALENs have been successful in shifting mtDNA heteroplasmy, but they all have drawbacks as gene therapy reagents, including: large size, heterodimeric nature, inability to distinguish single base changes, or low flexibility and effectiveness. Here we report the adaptation of a gene editing platform based on the I-CreI meganuclease known as ARCUS®. These mitochondrial-targeted meganucleases (mitoARCUS) have a relatively small size, are monomeric, and can recognize sequences differing by as little as one base pair. We show the development of a mitoARCUS specific for the mouse m.5024C>T mutation in the mt-tRNAAla gene and its delivery to mice intravenously using AAV9 as a vector. Liver and skeletal muscle show robust elimination of mutant mtDNA with concomitant restoration of mt-tRNAAla levels. We conclude that mitoARCUS is a potential powerful tool for the elimination of mutant mtDNA.

scholarly journals CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

2021 ◽  
Author(s):  
Fiona M Wilson ◽  
Richard J Harrison
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Gene Editing ◽  
Polyketide Synthase ◽  
Marker Gene ◽  
Constitutive Expression ◽  
Cost Effective ◽  
Editing Efficiency ◽  
Base Editing ◽  
Fusarium Venenatum

AbstractBackgroundGene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in a number of filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™).ResultsWe present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0-40% of isolates, with evidence of off-target editing. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum.ConclusionsUsing an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum, such as modulation of gene expression, gene insertion, base editing and prime editing. These tools will facilitate an understanding of the controls of secondary metabolism and hyphal development during fermentation of F. venenatum for mycoprotein production and may be used to validate prototypes of strains for improvement using classical means, enabling more cost-effective and sustainable production of this industrially important fungus.

scholarly journals CRISPR/Cas9 mediated editing of the Quorn fungus Fusarium venenatum A3/5 by transient expression of Cas9 and sgRNAs targeting endogenous marker gene PKS12

2021 ◽  
Author(s):  
Fiona Wilson ◽  
Richard J Harrison
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Gene Editing ◽  
Polyketide Synthase ◽  
Marker Gene ◽  
Constitutive Expression ◽  
Cost Effective ◽  
Editing Efficiency ◽  
Base Editing ◽  
Fusarium Venenatum

Abstract Background Gene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in a number of filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™). Results We present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0–40% of isolates, with evidence of off-target editing. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum. Conclusions Using an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum, such as modulation of gene expression, gene insertion, base editing and prime editing. These tools will facilitate an understanding of the controls of secondary metabolism and hyphal development during fermentation of F. venenatum for mycoprotein production and may be used to validate prototypes of strains for improvement using classical means, enabling more cost-effective and sustainable production of this industrially important fungus.

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